The present exemplary embodiment relates to cell screening techniques. It finds particular application in conjunction with cell screening methods and related instrumentation, and will be described with particular reference thereto. However, it is to be appreciated that the present exemplary embodiment is also amenable to other like applications.
Many medical applications would benefit from the ability to detect rare cell events. For example, it is known that fetal cells circulate in the maternal blood stream. See Oosterwijk et.al., Am. J. Hum. Genet., 63: 1783-1792 (1998); Bajaj et. al., Cytometry, 39: 285-294 (2000), the contents of which are incorporated by reference herein. Cancer cells can be found circulating in the bloodstream depending on the stage of cancer. See Hochten-Wollmar et. al., Int. J. Cancer, 70: 396-400 (1997), the contents of which are incorporated by reference herein. Shed tissues of people infected with viruses can also be detected in the circulation system at an early stage. See Musiani et. al., J. Histochem. Cytochem., 45(5): 729-735 (1997), the contents of which are incorporated by reference herein. Early detection of these rare cells would allow invasive diagnostic procedures such as amniocentesis to be avoided, cancer development to be properly monitored and treatment to be prescribed, or viral outbreak to be prevented. Detection of these rare cells would also find use in other diagnostic or research applications.
To provide statistically significant information, it is necessary to screen about 50-100 million blood cells in order to detect rare cell events occurring at scales of 1 in 1 to 10 millions. Therefore, a system that could efficiently and quickly process a large number of cells, such as up to 100 million, at a time would be beneficial. However, these numbers are exemplary and should not be construed as limiting.
Measuring emitted light has become a widely used method in detecting rare cell events. Two methods may be used to create these emitted photons. Chemiluminescence (CL) refers to the light emitted by a chemical reaction, especially when the chemical reaction is catalyzed by an enzyme. For example, luciferase catalyzes the oxidation of luciferin and produces green light. Alkaline phosphatase can be used with 1,2-dioxetane substrates such as CSPD and CDP-Star to produce light as well. In one method, the enzyme is linked to an antibody specific to a protein marker and the antibody binds the protein. Reagents are introduced which are cleaved by the enzyme and cause CL. A second analytical technique known as fluorescence uses a molecule (usually an organic dye or fluorchrome but more recently inorganic semiconductor nanocrystal material such as quantum dot is also used as a tagging molecule) which absorbs light at one wavelength and emits light at a different wavelength. The molecule is first bound to a cell or cell component such as a protein, lipid, chromosome, or other such components. A light (usually a laser) is then used at the wavelength absorbed by the molecule to excite it and the amount of light emitted at the emission wavelength is measured. These assays are useful because the amount of light emitted is proportional to, and thus can be used to determine the number of cells or cell components to which the molecule is bound that is present in the sample. Similarly, the rate of light output can also be used to determine the concentration of the cells or cell components present in the sample. These assays are very sensitive, have a wide dynamic range within which they can be used, and have low background noise. The kinetics of CL also allow for two different types of kinetics. In “flash” kinetics, light output peaks rapidly, then dies off quickly. In “glow” kinetics, light output is steady for a comparatively long period of time. Because of these characteristics, they are widely used in studying gene expression and regulation within living cells as well as for protein/nucleic acid blots.
Cell detection based on illumination to generate fluorescence is currently used in two main categories: flow cytometry and, image cytometry, which includes conventional or digital microscopy as well as laser scanning microscopy. In flow cytometry, cells are suspended in solution and travel one by one past a sensing point. Fluorescent compounds can be attached to the cells or cell components and detected using a laser which excites the compound and causes it to fluoresce. In conventional fluorescent microscopy, cells are fixed on a slide and stained with a fluorescent compound, then viewed under a microscope. Broad spectrum light sources (such as mercury arc lamp or Xenon flash lamp) coupled with specific excitation/emission filters are used to block undesired excitation lights and allow detection of weaker fluorescence. They usually include automatic stage movement and use low-magnification scanning to identify potential cells of interest followed by high magnification to reject false positives. Bajaj described one such setup of a fluorescent microscope. It's also noted that laser can be used to excite these fluorescent compounds in a system called laser scanning cytometer (LSC). Both of these image-based cytometry methods direct the excitation light and collect the fluorescence through a microscope objective.
However, both flow cytometry and fluorescent microscopy have several problems which hinder their use in clinical applications. Bajaj et al. concluded that flow cytometry could be used to screen cells quickly, but generated high numbers of false positives due to autofluorescence, nonspecific staining, and cell aggregates. See also Radbruch et. al., Curr. Opin. Immunol. 7:270-273 (1995), the contents of which are incorporated by reference herein. Also, in practice a high number of cells bunch or clump together, making it impossible to examine each cell separately. The cells being examined cannot be saved for confirmation of the diagnosis, nor can the high-resolution images needed for such confirmation be taken by current instrumentation.
Conventional fluorescent microscopy or laser scanning cytometry are unsuitable for clinical use because it usually requires at least 10-20 microscope slides and from 5 to 30 hours to scan 100 million blood cells. In addition, the use of fluorescence creates the need to filter strong exciting light and also leads to the possibility of bleaching, both of which affect the sensitivity of the assay. However, microscopy allows the cells to be saved for confirmation or for images to be captured either digitally or on film. Finally, the equipment costs of both techniques are very high.
A significant cause of delay in imaging is related to the process by which cells or samples are examined. In current image cytometry systems, the cell samples are usually processed in two steps. First, the imaging device (e.g., a microscope with a CCD camera) is used at low resolution and passes over the entire area of the slide in order to detect all “potential hits.” However, with a typical field of view of only a few square millimeters through a low-magnification 4× objective and a CCD camera, moving the objective across entire surfaces of multiple slides is a very time-consuming bottleneck. After this “pre-screening,” the imaging device then reexamines the “potential hits” at high resolution (such as 20× or 40×) by making a second pass over the slide. This second pass increases the amount of time required for a complete examination of the cell samples.
There is therefore a need for methods and systems which are less expensive, have higher throughput of cells, and allow for confirmation of results. The present exemplary embodiment contemplates a new and improved approach for rare cell detection methods and systems which overcome the above-referenced problems and others.